KR20010113088A - Novel mouse CIA protein and CIA gene having anti-apoptotic activity as a selective inhibitor of CAD interacting with ASK1 and use thereof - Google Patents

Abstract

The present invention relates to novel CIA (CAD inhibitor interacting with ASK1) proteins, CIA genes and their use, which specifically inhibit apoptosis, and specifically to isolate them from mice by yeast 2-hybrid screeening. A novel CIA protein, a CIA gene, that binds to caspase activated-DNase (CAD / DFF40 / CPAN) and apoptosis signal-regulating kinase ASK1 (ASK1) to inhibit apoptosis And uses thereof. The novel mouse CIA protein or CIA gene of the present invention can be used as a selective inhibitor against CAD and ASK1 mediated apoptosis or for the treatment, prevention, diagnosis or research of degenerative brain neurological diseases such as dementia, stroke, immune system diseases, etc. It can be usefully used for the process.

Description

Novel mouse CIA protein and CIA gene having anti-apoptotic activity as a selective inhibitor of CAD interacting with ASK1 and use according as selective inhibitors of CAD to bind AS1 }

The present invention relates to novel CIA (CAD inhibitor interacting with ASK1) proteins, CIA genes and their use, which specifically inhibit apoptosis, and specifically to isolate them from mice by yeast 2-hybrid screeening. A novel CIA protein, a CIA gene, that binds to caspase activated-DNase (CAD / DFF40 / CPAN) and apoptosis signal-regulating kinase ASK1 (ASK1) to inhibit apoptosis And uses thereof.

Apoptosis or programmed cell death (PCD) is the basic intracellular process for spontaneous self-elimination of damaged or unwanted cells in a wide range of biological systems, including organ development, tissue remodeling. It is an essential mechanism for formation, maintenance of intracellular homeostasis and removal of abnormal and damaged cells. As such, the apoptosis process, which plays an important role in the organism, must be precisely regulated according to a defined program. If apoptosis is improperly progressed, cancer, autoimmune disease, and various neurodegenerative diseases Leading to diseases such as Kerr et al., Br. J. Cancer , 26, 239-257, 1972. Early cells undergoing apoptosis develop chromatin aggregation, cytoplasm, and the like during late stages of the disease, such as damage to cell junctions, blebbing of cell membranes, and cell shrinkage. Nuclear enrichment (cytoplasm and nuclei condensation), loss of mitochondrial membrane potential, changes in plasma membrane composition, and apoptotic body formation are induced to undergo morphological and biochemical changes throughout. DNA fragmentation in the form of oligonucleosomes, the final result of apoptosis, is a biochemical feature of cells undergoing apoptosis (Green DR, and Reed, JC, Science , 281, 1309-1312, 1998 ).

Many researchers have now identified key players and mechanisms involved in apoptosis in a variety of organisms. In nematodes such as Carnorhabdtis elegans , three molecules of CED-3, CED-4 and CED-9 have been reported to play an important role in regulating apoptosis mechanisms that occur during nematode development. Both CED-3 and CDE-4 are essential for inducing apoptosis, while CED-9, which shows homology between the Bcl-2 family and amino acid sequence in mammals, acts identically to Apaf-1 in mammals. Acts as an inhibitor of CED-4. In particular, CED-3 is known to be involved in apoptosis with homology with caspase, a kind of cellular cysteine protease in mammalian cells.

Caspases are activated by various stimuli that induce apoptosis, resulting in poly (ADP-ribose) polymerase (RARP), lamine, cytokeratin and caspase. -Induces apoptosis formation by degrading cellular proteins such as activated DNase inhibitors (ICAD) (Cryns, V., and Yuan, J., Genes Dev ., 12, 1551-1570, 1998). Cascades include receptor-mediated signal transduction, depletion of growth factors, oxidative stress, DNA damage, and cell-cells or cells. It can be activated under a variety of conditions, including disruption of cell-matrix interaction bonds, to induce apoptosis. Active research has shown that in mammals, at least 14 different classes of caspases so far are peptide substrates after aspartic acid residues, in which the cascades play a pivotal role in the initiation and execution of apoptosis. Has been reported to induce apoptosis by cleaving (Nunez et al., Oncogene, 17, 3237-3245, 1998).

In addition, caspase activity is very closely associated with nuclear DNA fragmentation, one of the most important and irreversible events in apoptosis. The DNA of cells undergoing apoptosis is cleaved by endogenous endonuclease to form a large number of DNA fragments of about 180 to 200 bp, and these DNA fragments are electrophoresed on an agarose gel to identify a ladder. It shows a ladder-like pattern. CAS (caspase activated DNase), which plays the same role as the endonuclease, is also called caspase-activated nuclease (CPAN) and DNA fragmentation factor (DFF40). Isolated from mice (Liu et al., Proc. Natl. Acad. Sci. USA ., 95, 1841-1846, 1998; Liu et al., Cell, 89, 175-184, 1997). CAD / DFF40 originally had intrinsic endonuclease activity, but in growing cells it is inactivated in combination with its inhibitor, ICAD. Thus, full length form (ICAD-L) of 331 amino acids, which is combined with CAD and suppresses the endonuclease activity of CAD, is separated from human HeLa cells and is a 45 kDa DNA fragment. Known as the mouse counter part of the DFF45, which is called the fire factor. In addition, short length form (ICAD-S) consisting of 1-261 amino acids of ICAD-L and four C-terminal amino acids specific to ICAD-S is used in mouse lymphoma cells. Expression has been reported (Sakahira et al., J. Biol. Chem ., 274, 15740-15744, 1998).

ICAD / DFF45 not only acts as a selective inhibitor of CAD / DFF40, but also acts as a molecular chaperone to induce proper folding of CAD / DFF40 protein. In general, CAD / DFF40 is inactivated in combination with the inhibitor ICAD / DFF45, and cascade-3 cleaves two positions of Asp117 and Asp224 of ICAD / DFF45 to inactivate ICAD / DFF45 and CAD / DFF40. Has endonuclease activity (McIlroy et al., Oncogene, 18, 4401-4408, 1999). Of the many caspases that have been discovered to date, caspase-3 and caspase-7 are known to inactivate ICAD / DFF45 by cleavage in vitro. However, it has been reported that caspase-7 can cleave and inactivate ICAD / DFF45, but cannot form active CAD / DFF40, making caspase-3 a major activator of CAD-dependent DNA fragmentation. (Wolf et al., J. Biol. Chem. , 274, 30651-30656, 1999). In addition to ICAD / DFF45, CIDEs contain N-terminal CIDE-N domains homologous to the conservative CIDE-N of CAD / DFF40 and ICAD / DFF45, suggesting that CIDEs proteins are involved in CAD / DFF40 activity. Inohara et al., Embo. J. , 17, 2526-2533, 1998).

Mitogen-activated protein kinase (MAPK) signaling, on the other hand, is closely associated with apoptotic cell death (Cobb, MH, and Goldsmith, EJ, J. Biol. Chem., 270, 14843-). 14846, 1995; Dickens et al, Science , 277, 693-696, 1997). MAPK signaling is known to regulate the expression of numerous genes that are activated by extracellular stimuli and are involved in apoptosis and survival. Protein kinases involved in MAPK signaling are divided into three different components: MAPKs, MAPK kinase (MAPKKs) and MAPK kinase kinase (MAPKKKs). Looking at their mechanism of action, MAPKKKs activated by the kinase or conjugate protein in the upper position of the protein kinase induces the activity by phosphorylating MAPKKs, and thus induced MAPKKs induced to activate MAPKs again. Active MAPKs then move into the nucleus to regulate gene expression or other cellular events by activating various transcription factors or kinase in lower positions.

Signaling mechanisms associated with apoptotic apoptosis, such as MAPK signaling, have revealed at least three different signaling pathways in mammalian cells. Extracellular signal-regulated kinase (ERK), c -jun N-terminal kinase / stress-activated protein kinase (c-jun N-terminal kinase / stress-activated protein kinase) and p38 MAPK (Boulton et al., Cell, 65, 663-675, 1991; Cobb , MH, and Goldsmith, EJ, J. Biol. Chem., 270, 14843-14846, 1995; Frnger et al., Curr. Opin. Genet. Dev. , 7, 67-74, 1997; Han et al., Science, 265, 808-811, 1994).

The ERK pathway is primarily activated by mitogenic growth factors such as insulin, epidermal growth factor, and phorbol esters, leading to growth factor-mediated activity and growth factor. -mediated activation and differentiaons. In contrast, the JNK pathway is preferentially activated by invasive pressure and thermal stimuli, environmental stress stimuli such as UV and proinflammatory cytokines (Ahmed, A., and Salahuddin, A., Indian. J. Biochem. Biophys. , 31, 156-159, 1994; Derijard et al., Science , 267, 682-685, 1994; Galcheva-Gargova et al., Science , 265, 806-808, 1994), to HOG1 kinase found in yeast P38 kinase, which shows very high homology, is induced by hyperosmotic pressure, lipopolysacharide, tumor necrosis factor α and interleukin-1 (Han et al., Science 265, 808-811, 1994; Rouse et al., Cell , 78, 1027-1037, 1994).

In particular, the JNK and p38 pathways have been suggested to play an important role in apoptotic apoptosis induced by various stresses (Butterfield et al., J. Biol. Chem ., 272, 10110-10116, 1997). Kawadaki et al., J. Biol. Chem. , 272, 18518-18521, 1997). To date, three jnk genes, jnk1, jnk2 and jnk3 have been isolated (Gupta et al., Embo. J. , 15, 2760-2770, 1996; Widmann et al., Physiol. Rev. 79, 143-180, 1999), the jnk genes include two MAPKKs, MKK4 / SEK1 (Derijard et al., Cell, 76, 1025-37, 1999) and MKK7 (Holland et al., J. Biol. Chem. , 272, 24944-). 24948, 1997). p38 kinase was isolated from four genes p38a, p38b, p38c and p38d (Goedert et al. , Embo. J., 16, 3563-3571, 1997; Han et al., Science, 265, 808-811, 1994 Jiang et al., J. Biol. Chem. , 272, 30122-30128, 1997; Lee et al., Nature , 272, 23668-23674, 1994; Wang et al., J. Biol. Chem., 272, 23668-23674, 1997), which are activated by MKK3 and MKK6. In addition, various MAPKKKs have been isolated as upstream activators of the JNK and p38 pathways, of which tumor growth factor-activated kinase (TAK1) and MEKK4 / MTK1 are responsible for both JNK and p38 pathways. Activating (Gerwins et al., J. Biol. Chem., 272, 8288-8295, 1997; Takekawa et al., Embo. J. , 16, 4973-4982, 1997; Yamaguchi et al., Science, 270, 2008-2011, 1995), MAPKKK, apoptosis signal-regulating kinase 1 (ASK1), has also been reported to activate both JNK and p38 pathways (Ichijo et al., Science, 275, 90-94,1997). Yamaguchi et al., Science, 270, 2008-2011, 1996).

On the other hand, ASK1 is a type of MAPKKK and selectively activates the SEK1-JNK and MKK3 / 6-p38 pathways. Previous reports indicate that ASK1 plays an important role in TNFα- and Fas-induced apoptosis (Hsu et al., Immunity, 4, 387-396, 1996; Rothe et al., Cell , 83, 1243). -1252, 1994; Shu et al., Proc. Natl. Acad. Sci. USA , 93-13973-13978, 1996). ASK1 is involved in cellular mechanisms that can regulate apoptosis induced by a variety of stresses, including TNFα and Fas, as well as H ₂ O ₂ , ultraviolet light and other DNA-induced agents. ASK1, which is involved in TNFα- and Fas-induced apoptosis, is known to induce apoptosis by activating the C-terminal noncatalytic region of ASK1 in combination with the conservative C-terminal TRAF domain of TRAF2, a TNF-receptor (Hoeflich). et al. , Oncogene , 18, 5814-5820, 1999). In addition, ASK1 can bind to Daxx, which is required for Fas activity (Chang et al., Science, 281, 1860-1863, 1998). This Daxx-induced ASK1 activation is associated with JNK. all of the p38 pathways lead to apoptotic cell death (Chang et al., Science , 281, 1860-1863, 1998; Yang et al., Cell, 89, 1067-1076, 1997). Thus, since ASK1 plays an important role in cytokine-induced apoptosis as mediators of TNFα- and Fas-induced intracellular signal cascades, studies on the proteins and molecular mechanisms regulating ASK1 activity It is very important to understand the apoptosis process.

Therefore, the present inventors have studied to find a protein that can physically bind to ASK1, and isolated a novel ASK1-binding protein CIA (CAD inhibitor interacting with ASK1), a CAD inhibitory protein from mice. We not only inhibit apoptosis by ASK1-induced apoptosis by inhibiting stress- or cytokine-induced ASK1 activity by binding CIA directly to ASK1, but also by inhibiting DNA fragmentation induced by CAD by binding to CAD. The present invention was completed by confirming the inhibition both in vivo and in vitro. Thus, the novel mouse CIA proteins or CIA genes of the present invention can be used as selective inhibitors of apoptosis mediated by CAD and ASK1 or in the treatment, prevention, diagnosis or treatment of degenerative brain neurological diseases such as dementia, stroke, immune system diseases or the like. It can be usefully used for the research process.

It is an object of the present invention to provide a novel substance capable of modulating apoptosis activity caused by various kinds of stress.

In order to achieve the above object, the present invention provides a mouse CIA protein and cDNA encoding the same.

In addition, the present invention using a mouse CIA gene or mouse CIA protein as a probe, a nucleic acid or protein interacting with the CIA protein; Analogs of the CIA gene; Or a method of searching for analogs of CIA proteins.

In addition, the present invention provides a gene therapy agent comprising a gene construct containing a mouse CIA gene as an active ingredient.

Finally, the present invention provides an inhibitor of apoptosis activity comprising mouse CIA protein and a therapeutic agent for apoptosis related diseases.

1 shows amino acid sequences of mouse CIA proteins and amino acid sequences of human and yeast showing homology thereto,

Figure 2 shows the distribution of tissue in mouse CIA transcripts using multiple tissue northern blot analysis,

3 shows the results of confirming that CIA is expressed from various mammalian cell lines and mouse brain tissues using western blot analysis using anti-CIA antiserum.

4 is a result confirming that the C-terminal portion of CIA binds to the N-terminal portion of ASK1 using in vitro binding analysis of CIA and ASK1 using various deletion mutants of CIA.

5 is a result confirming that the CIA inhibits the kinase activity of ASK1 using immunoprecipitation using an anti-Flag antibody,

6 shows that ectopic expression of CIA in 293 cells inhibits the kinase activity of ASK1,

7 is a result confirming that CIA inhibits the enzymatic activity of JNK induced by ASK1 using immunocomplex kinase analysis,

8 is a result of measuring luciferase activity (luciferase actuvuty) confirming that CIA inhibits the activity of c-JUN transcription factor induced by ASK1,

9 is a result confirming that the N-terminal portion of the CAD and the CIDE-N domain of CAD through in vitro binding analysis of CIA and CAD,

10 shows that CIA inhibits in vitro CAD nuclease activity,

11 is a result confirming that CIA is expressed in 293 cells to inhibit DNA fragmentation induced by caspase-3,

12 shows that CIA transfected with 293 cells is stably expressed in cells,

FIG. 13 shows that 293 cells transfected with CIA are resistant to apoptosis induced by various stresses,

FIG. 14 shows that 293 cells transfected with CAI are resistant to apoptosis induced by the activity of ASK1.

Hereinafter, the present invention will be described in detail.

The present invention provides a mouse CIA gene, homologs thereof, and variants thereof as described in SEQ ID NO: 3 . The present invention also provides a mouse CIA protein as described in SEQ ID NO: 4 or a variant thereof.

"Homologs" of the mouse CIA gene refers to a gene comprising a coding sequence for the mouse CIA protein set forth in SEQ ID NO: 4 . Thus, the category of homologous genes includes all kinds of genes capable of producing a mouse CIA protein as depicted in SEQ ID NO: 4 when expressed.

"Variants" of the mouse CIA protein refer to the deletion, addition, insertion or substitution of one or more amino acid residues of the mouse CIA protein set forth in SEQ ID NO: 4 . However, the variant protein may be at least 90% or more in part at a portion corresponding to amino acid residues 1 to 79 of the N-terminus of SEQ ID NO: 4 and at a portion corresponding to amino acid residues 174 to 221 of the C-terminus. It is defined as having homology and having a homology of at least 80% as a whole.

By “variants” of the mouse CIA gene is meant any gene encoding a variant protein of the mouse CIA protein.

In order to search for novel proteins that act as negative regulators of apoptosis, we use a yeast two-hybrid system using ASK1 to react with caspase activity-DNase. A novel mouse CIA gene encoding the ASK1 binding protein, a inhibitor activated-DNase inhibotor (CIA), was isolated. Since mice are frequently used for the production of transgenic animals and mutation studies by gene targeting, the mouse CIA gene of the present invention can be easily used for such studies.

To isolate novel proteins that can specifically bind ASK1 to induce apoptosis in mice, full-length ASK1 was used to search for 2 x 10 ⁶ phage plagues prepared from the mouse neural cDNA library. By selecting a novel mouse cDNA clone encoding a protein of 0.9 Kb size and confirmed that the clone specifically reacts with ASK1.

In order to obtain a complete mouse cDNA clone in the 5 'region, a 5'-rapid amplification of cDNA ends (5'-RACE) was used as a template for the cranial nervous system cDNA of the mouse, thereby obtaining a 930 bp CIA cDNA clone and We have included this complete open reading frame (ORF). The mouse cDNA clone was named CAD inhibitor interacting with ASK1, ie CIA ( SEQ ID NO: 3 ). The CIA cDNA clone consists of 920 nucleotide sequences set forth in SEQ ID NO: 3 , including an ORF with 666 nucleotides, and the amino acid sequence obtained therefrom consists of 221 amino acids set forth in SEQ ID NO: 4 . Sequencing revealed no in-frame dtop codons in the 5'-untranslated region of mouse CIA.

Human CIA clones were isolated using RT-PCR based on the amino acid sequence of mouse CIA protein. The human CIA gene is described by SEQ ID NO. 7 , and the amino acid sequence of SEQ ID NO. 8 obtained therefrom is compared with the mouse CIA amino acid sequence, and showed at least 99% homology with each other (see FIG. 1 ). A comparison of the amino acid sequences of mouse CIA using the BLAST Program revealed 35% homology with the putative protein of Arabidopsis thaliana , and the Sachharomyces serevisiae VPS28 ( Sachharomyces serevisiae). VPS28) and Schizoachharomyces pombe VPS ( Schizoachharomyces pombe VPS) showed about 28% homology and did not have any protein domain motifs reported so far (see FIG. 2 ).

In addition, in the present invention, using the CIA gene, homologous gene, mutant gene, mouse CIA protein, or mutant protein of the mouse as a probe, a nucleic acid or protein interacting with the CIA protein, analogue of the CIA gene ( analogs) or methods for searching for analogs of CIA proteins.

The term “analogue” refers to a gene or protein that exhibits at least 80% sequence homology at a randomly selected 30 bp or 10 amino acid residue when compared to the CIA gene set forth in SEQ ID NO: 3 or the CIA protein set forth in SEQ ID NO: 4 . In addition, the expression "interacting" means a state capable of physical binding directly or indirectly to the CIA protein. The term "probe" refers to conventional Southern blot analysis, Northern blot analysis, Western blot analysis, far Western blot analysis, gel mobility shift analysis, library screening , Probes that can be used for affinity chromatography, etc., as well as probes that can be used for DNA chips using automated devices, and specific primers used for polymerase chain reaction (PCR). That is, it refers to a probe used in all assays that use, in principle, the interaction between nucleic acid-nucleic acid, protein-protein, and protein-nucleic acid. The probe may be used by labeling it with radioisotopes, fluorescent substances, or the like.

Therefore, the search method of the nucleic acid or protein is carried out according to the analysis method or the search method presented above. For example, an analog of the CIA gene can be cloned by performing PCR using some specific sequences of the CIA gene of the present invention and searching the cDNA library using the DNA fragments obtained as a probe again. As another example, the CIA protein of the present invention can be immobilized on the resin of an affinity chromatography column to search for a protein and the like that can physically bind to the CIA protein under specific conditions.

The present invention also provides a gene therapy agent comprising a gene construct containing a mouse CIA gene as an active ingredient.

The gene construct is a means for efficiently expressing a CIA gene, a homologous gene thereof, or a variant gene thereof included therein, and may modify its components according to a specific purpose of gene therapy. For example, when performing gene therapy for the purpose of increasing or decreasing the expression of endogenous CIA genes, the gene construct is prepared by including a transposon sequence for homologous recombination around the CIA gene. can do. As another example, in order to efficiently express exogenous mouse CIA gene, a gene construct prepared by connecting a strong promoter to the CIA gene, homologous gene or mutant gene of the mouse may be used. On the other hand, the construction of the gene construct can be modified according to the specific gene therapy means, for example, when performing gene therapy using a recombinant virus, using a known vector derived from a viral genome such as retrovirus The gene therapy agent of the present invention can be constituted.

The gene therapy agent is caused by an inadequate regulation of apoptosis processes such as cancer, degenerative neurological disease, stroke, immune system disease, and inflammation, as well as treatment of diseases caused by inadequate regulation of apoptosis activity due to inherited or acquired genetic defects. It can be usefully used for the treatment of diseases. In the present invention, by confirming that the CAD and ASK1 activity is lowered in the mammalian cell line expressing the CIA gene, it was confirmed that the gene therapy agent of the present invention is useful for the disease.

The present invention also provides inhibitors for selectively inhibiting apoptosis activity by CAD and ASK1, including mouse CIA proteins, and therapeutic agents for apoptosis-related diseases.

Since the inhibitor of CIA activity includes mouse CIA protein or its mutant protein as an active ingredient, it may selectively inhibit apoptosis activity by CAD and ASK1, and selectively inhibit signaling processes involving CAD and ASK1 activity. It can also be used for the purpose.

The apoptosis-related diseases are diseases caused by abnormal control of apoptosis processes, and examples thereof include cancer, degenerative neurological diseases, stroke, immune system diseases, and various inflammations.

The inhibitor of CIA activity and the treatment for apoptosis-related diseases include the CIA protein described in SEQ ID NO: 4 or a variant protein thereof as an active ingredient.

The inhibitor and the therapeutic agent may be administered orally or parenterally during clinical administration, and parenteral injection administration is particularly preferred. That is, the inhibitor and the therapeutic agent may be administered in various oral or parenteral dosage forms during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used. Is prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, or CIA protein or a variant protein thereof. , Sucrose (Sucrose) or lactose (Lactose), gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The effective dose of the mouse CIA protein and its variant protein is 1-10 mg / kg, preferably 1-5 mg / kg.

In the embodiment of the present invention, by identifying the biological properties of the CIA gene and CIA protein of the mouse was confirmed the preferred use of the gene and protein.

The present inventors have found that CIA, which is an ASK1-binding CAD inhibitor protein, directly binds to the N-terminus of ASK1 in both in vitro and in vivo to inhibit ASK1 activity as well as acting as a CAD inhibitor that binds directly to CAD. Inhibition of cell death and DNA fragmentation by apoptosis was confirmed to act as an anti-apoptotic protein that inhibits the activity of ASK1 early in apoptosis and later inhibits CAD nuclease activity.

Specifically, the inventors performed a yeast two-hybrid screening method to find a protein that modulates the activity of ASK1 by physical binding, and selected CIA clones that bind ASK1 from the mouse brain cDNA library. Expression of the mouse CIA gene was confirmed by mouse multi-tissue Northern blot analysis. As a result, transcripts of CIA were detected in various mouse organs such as heart, brain, liver, and testis (see FIGS . 2 and 3 ).

In vitro binding analysis of CIA and ASK1 using the GST-fusion deletion mutant of CIA revealed that CIA has a stress- or cytokine-induced ASK1 activity by physically binding its C-terminus to the N-terminus of ASK1. Was inhibited (see FIG. 4 ).

In addition, CIA acted as an inhibitor of ASK1 that inhibits the kinase activity of ASK1 induced by stress such as UV or TRAF2 in both in vivo and in vitro experiments (see FIGS . 6 and 7 ), and ectopic expression of CIA. (ectopic expression) also inhibited the expression and kinase activity of signal regulatory proteins such as JNK1 in the subregion of ASK1 by inhibiting the enzymatic activity and expression of ASK1 (see FIGS . 8 and 9 ).

In addition, the present inventors performed a yeast two-hybrid screening method to isolate proteins capable of physically binding to CAD involved in DNA fragmentation. As a result, the positive clones isolated from mouse and human brain cDNA libraries were identified. The ASK1-reactive protein of CIA has the same base sequence as the isolated CIA, and the CIA of the present invention, which acts as an inhibitor of ASK1 activity, physically reacts with CAD involved in DNA fragmentation to regulate apoptosis. Confirmed that it can.

In vitro binding analysis of CIA and CAD using a deletion mutant of CIA revealed that CIA protein binds directly to the N-terminal CIDE-N domain conserved within CAD / DFF40 and CIDE through its N-terminal site. Activity was inhibited (see FIG. 10 ).

In addition, in vivo and in vitro experiments showed that intermolecular binding of CIA to CAD inhibited the nuclease activity of CAD and inhibited DNA fragmentation induced by caspase-3 active CAD (see FIGS . 11 and 12 ). ). The N-terminal CIA fragment, which lacks the binding site of CAD, did not inhibit the nuclease activity of CAD, which was obtained by direct protein-protein interaction. It is displayed.

CIA was transfected into mammalian cells and examined for their expression. CIA was stably expressed in mammalian cells, and actually inhibited the activities of ASK1 and CAD, which are induced by various stresses and activators of the JNK / SAPK pathway. Apoptosis was inhibited ( FIGS. 13 and 14 ). These results suggest that CIA is an anti-apoptotic gene product that can regulate JNK / SAPK signaling, which is activated early in apoptosis, as well as DNA fragmentation by the nuclease activity of CAD resulting in late apoptosis.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Screening of Mouse CIA Protein Specific to ASK1

<1-1> yeast two-hybrid screening

In order to isolate mouse proteins that can specifically bind ASK1 causing apoptosis, we performed a yeast double-hybrid screening method using full length ASK1.

Specifically, the full-length ASK1 cDNA is fused to the LexA DNA binding domain in the pLexA bait plasmid (Clonetech), and the fusion construct is EGY48 [p8op-lacZ] ( Saccharomyces cerevisiae) in the yeast Saccharomyces cerevisiae. EGY48 [p8op-lacZ], Clonetech). About 2 × 10 ⁶ clones were screened by searching for a mouse brain Lambda ZAPII cDNA library (Stratagene) cloned in pB42AD prey plasmid (Clonetech) as the yeast transformant. CIA cDNA clones isolated by yeast two-hybrid screening were probed by labeling ³² P radioisotopes by a random priming method using a Prime-It II random primer labeling kit (Stratagene) Used as. Filters taken from mouse brain cDNA library phage plaques were placed in a prehybridization buffer (5 × SSPE, 5 × Denhardt's solution, 0.15% SDS, 50% formamide and 100 μg / ml denatured salmon sperm DNA) at 60 ° C. for 2 hours. Soak for and hybridize with the probe at 42 ° C. for at least 12 hours. The filter was washed and then developed on an X-ray film using an intensifying screen at -70 ° C to separate positive phage clones. The positive cDNA insert thus isolated was cloned into the restriction enzyme EcoRI site of pBluescript SK (-) (Stratagene) phagemid in vivo to analyze the nucleotide sequence.

As a result, the inventors obtained a CIA cDNA encoding 0.9 kb of protein from the cDNA library of the mouse brain, and performed Northern blot analysis on the whole RNA isolated from the adult mouse brain using the cDNA clone as a probe. It was confirmed that about 1.0 kb of hybridized mRNA band was detected.

<1-2> 5'-RACE

In order to obtain CIA cDNA in the 5 'region, the inventors performed 5'-RACE (5'-rapid amplification of cDNA ends, Boeriger Menheim) using the cDNA clone as a template for the whole mRNA isolated from mouse brain tissue. It was. At this time, the primers were CIA-GSP described in SEQ ID NO: 1 and CIA-Bam described in SEQ ID NO: 2 , and the PCR product obtained using the primer set was cloned into pBluescript SK (-) plasmid and analyzed for nucleotide sequence. It was.

As a result, the present inventors obtained a CIA cDNA clone of 930 bp and confirmed that the clone contained a complete open reading frame (ORF). The clone consists of 920 nucleotide sequences as set forth in SEQ ID NO: 3 having an ORF of 666 bp, and confirmed that there was no in-frame stpo codon in the 5'-untranslated sequence. . Analysis of the ORF included in the cDNA sequence sequence ORF encoded a mouse CIA protein consisting of 221 amino acids described in SEQ ID NO: 4 .

<1-3> Isolation of Human CIA Clones

In order to isolate human CIA clones as a counterpart of mouse CIA, the present inventors have identified CIA-H2 primers as set forth in SEQ ID NO: 6 and CIA-H1 primers as set out in SEQ ID NO: 6 from EST clones having a very high homology to the nucleotide sequence. Primer was prepared. As a result of analyzing the nucleotide sequence by separating the human CIA clone from the whole RNA of the human fetal brain through RT-PCR using the primer set, it was confirmed that it was composed of 913 nucleotides described in SEQ ID NO: 7 . The amino acid sequence of the human CIA protein described in SEQ ID NO: 8 obtained therefrom was compared with the amino acid sequence of the mouse CIA protein, and showed at least 99% homology with each other ( FIG. 1 ).

<1-4> Amino Acid Sequencing of Mouse CIA

Analysis of the amino acid sequence of mouse CIA described in SEQ ID NO: 4 by blast program shows that mouse CIA has 35% homology with the amino acid sequence of putative protein of Arabidopsis thaliana Vis showed 28% homology with VPS28 ( Saccharomyces cerevisiae VPS28) and Schizosaccharomyces pombe putative VPS ( Saccharomyces cerevisiae VPS28) and Schizosaccharomyces pombe putative VPS . 1 ).

Example 2 Expression and Distribution of Mouse CIA Gene in Tissues

<2-1> Multiple Organization Northern Blot Analysis

In order to investigate the expression pattern of the mouse CIA gene isolated from Example 1, multiple tissue northern blot analysis (Clontech) was performed.

Specifically, mouse CIA cDNA was labeled with ³² P radioisotopes by a random priming method using a Prime-It II random primer labeling kit, and then used as a probe to isolate poly (A) ⁺ RNA ( Hybridizations were performed on mouse multi-tissue blots containing 2 μg each).

As shown in FIG . 2 , about 1 kb of mouse CIA mRNA transcript is specifically strongly expressed in various tissues including heart, brain, liver and kidney, whereas the expression level in spleen, lung and muscle tissues is increased. Very low.

<2-2> Western Blot Analysis

We performed western blot analysis using anti-CIA anti-serum to confirm the expression level of CIA protein.

Specifically, after subcloning the mouse CIA cDNA obtained in Example 1 to pTrcHisC (Invitrogen), the expression vector was transformed into E. coli DH5α to express a His6-tagged CIA protein. The polyclonal antibody to anti-CIA-anti-CIA against anti-HIS 6-labeled protein was obtained by immunization by separating and purifying the heat-labeled CIA protein of 29 kDa size from the E. coli transformant. Cell lysate fractions (aliquots, 30 μg of protein) obtained from the brains of mice and various mammalian cell lines were electrophoresed and then subjected to Western blot analysis using the anti-CIA antibodies.

As a result, CIA protein with molecular weight of about 25 kDa was detected from various mammalian cell lines including mouse brain tissue and L929, NIH3T3, H4, HeLa, COS-7 and the like ( FIG. 3 ).

Example 3 In Vitro Binding Assay of CIA and ASK1

To investigate which position of the CIA protein binds to ASK1, we prepared GST-fusion recombinant proteins of deletion mutants of various CIAs.

Specifically, to prepare wild type of CIA and deletion mutants thereof, CIA-WT, CIA-ΔN, CIA-ΔC, and CIA-CENs, CIA-WT in full-length form, CIA-ΔN having N-terminus amino acids 1 to 79 deleted, CIA-ΔC having C-terminal amino acids 174 to 221 deleted, and both ends having been deleted from 79 DNA fragments encoding CIA-CEN containing only up to 174 amino acids were amplified by polymerase chain reaction (PCR), and then cloned into the vector pGEX-4T-1 (Pharmacia). By doing so, an expression vector for each DNA was prepared. GST-tags were made by connecting GST residues to the ends of the DNA fragments C. Then, each of the expression vectors was transformed into Escherichia coli BL21 (DE3), and the E. coli transformants were added in a medium to which 1 mM IPTG was added. The culture was induced to express the recombinant protein for each DNA fragment, and then the culture was removed by centrifugation. Transformant cells for each derivative from which the culture was removed were subjected to cell lysis buffer (cell lysis buffer, 50 mM Tris-HCl, pH 8.0), 100 mM EDTA, 1% Triton X-100, 1 mM PMSF and 0.5 mM After suspension in DTT), the cells were disrupted by sonication. After repeating this process five times, the supernatant was separated by centrifugation, and the supernatant was separated and purified using glutathione-agarose.

On the other hand, ASK1 and its mutants, ASK1-WT, ASK1-ΔN having deleted amino acids N-terminals 1 to 649, and ASK1-deleted amino acids from C-terminal positions 936 to 1375 △ C and after producing the recombinant proteins of the N- terminal region ASK1-NT containing the amino acid to the 656 times from one time as described above and labeled with ³⁵ S- methoxy Im methionine ⁽³⁵ S-methionine) in TNT retina particulate In vitro transcription was performed using the TNT reticulocyte lysate system (Promega).

GST-CIA-WT and its deletion mutant recombinant proteins isolated and purified as described above were immobilized on glutathione-agarose beads and then transcribed in vitro. [³⁵S] methionine-labeled ASK1 or mutant recombinant proteins of ASK1 were buffered (50 mM Tris, pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol, 0.1% NP-40 and 5 mg / ml BSA. Each CIA GST-fusion protein at 4 ° C. for 3 hours. After completion of the reaction, wash three times with wash buffer (50 mM Hepes [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Tween 20) and remove from each glutathione-agarose beads.³⁵S-labeled proteins were eluted and analyzed by SDS-polyamide gel electrophoresis.

As a result, the full-length wild-type recombinant protein CIA-WT and the recombinant protein CIA-ΔN deleted from the N-terminus were physically bound to ³⁵ S-labeled ASK1 transcribed in virto, but CIA-Δ deleted from the C-terminus. C, CIA-NT containing only the N-terminus and CIA-CEN lacking both ends did not interact with ASK1 ( FIG. 4 ).

In addition, CIA can bind to full-length ASK1, ASK1-NT containing only the N-terminus and ASK1-ΔC deleted from the C-terminus, but not ASK1-ΔN deleted from the N-terminus. ( FIG. 4 ).

Therefore, in order for CIA to physically bind with ASK1, it can be seen that the C-terminal region of CIA and the N-terminal region of ASK1 should interact with each other.

Example 4 Inhibitory Effect of CIA on ASK1 Kinase Activity

<4-1> Novel Inhibitor CIA of ASK1

In general, ASK1, which acts as MAPK kinase kinase (MAPKKK) in JNK / SAPK and p38 signaling, is known to be activated by cell exposure to various stresses. Therefore, the present inventors confirmed that the CIA directly binds to ASK1 in Example 3, and investigated whether CIA inhibits the kinase enzyme activity of ASK1.

Specifically, pcDNA3-ASK1-produced by cloning the cDNA clone of ASK1 (provided by Dr. Hidenori Ichijo of Tokyo University of Japan) to the EcoRI / XbaI restriction enzyme site of the pcDNA3-flag (Invitrogen) vector in 293 mammalian cell line. After transfection of the flag expression vector, ultraviolet light was irradiated at 60 J / m ² to induce kinase activity of ASK1. The transfected cells were treated with buffer A (20 mM Tris-HCl [pH 7.4], 150 mM sodium chloride, 1% Triton X-100, 1% deoxycholate, 12 mM β-glycerphosphate, 10 mM sodium florids, 5 mM EGTA and 1). Hemolysis using mM PMSF) was followed by centrifugation at 12,000 g for 15 minutes at 4 ° C. The precipitate was removed after centrifugation and the lysate obtained was immunoprecipitation using anti-Flag mouse monoclonal antibody (Boehringer Mannheim Inc.). Protein G-Sepharose ^TM beads (Protein G sepharose ^TM bead, Pharmacia) were added to the reaction solution, and then carefully mixed at 4 ° C. for 1 hour. The immunocomplexes obtained therefrom were washed three times with Buffer A, and the final immunoprecipitates were electrophoresed on SDS-polyamide gels to chemiluminescence detection system. Immunoblotting analysis was performed with mouse anti-Flag monoclonal antibody (Sigma) using Amersham. The activity of ASK1 was analyzed using GST-MKK6 (K82A) isolated in the presence of His-CIA.

As a result, when HIS-CIA recombinant protein was not added, phosphorylation of its substrate, GST-MKK6, was strongly induced by the kinase activity of ASK1 activated by ultraviolet light, whereas in the case where His-CIA recombinant protein was added, In vitro, the kinase activity of ASK1 activated by UV irradiation was inhibited, resulting in a significant decrease in phosphorylation of GST-MKK6 ( FIG. 5 ).

Thus, it can be seen that the CIA protein acts as a novel inhibitor of ASK1 without being phosphorylated by ASK1.

<4-2> Effect of Ectopic CIA on Intracellular ASK1 Activity

To investigate the inhibitory effects of CIA on ASK1 in mammalian cells, expression vectors encoding ASK1-flag, CIA and TRAF-2 (provided by Dr. David Goeddel of Tularik Inc., USA), a mammalian cell line 293 cells Were simultaneously transfected. 48 hours after transfection, the cells were not irradiated or irradiated with 60 J / m ² ultraviolet rays. The cells were lysed and immunoprecipitated using a mouse anti-Flag monoclonal antibody in the same manner as in Example <4-1>, and then immunocomplex kinase assay was performed to analyze the ASK1 activity of the immunoprecipitate.

As a result, the co-expression of CIA, as in the result of Example <4-1>, inhibited GST by inhibiting the kinase activity of ASK1 activated by UV irradiation or concentration-dependent expression of TRAF-2, a TNF receptor, in transfected cells. Phosphorylation of -MKK6 was significantly reduced ( FIG. 6 ).

Therefore, the results confirmed that CIA acts as an inhibitor of ASK1 both in vitro and in vivo.

<4-3> Inhibitory Effect of CIA on ASK1-mediated JNK / SAPK Activity

One of the major subregional signals of ASK1 is the activity of JNK / SAPK that promotes c-jun transcriptional activity. We investigated how CIA, which acts as an inhibitor of ASK1, affects JNK / SAPK activity. First, to investigate the effect of CIA on the kinase activity of JNK1 , each expression vector expressing ASK1, HA-SAPKβ (Shim et al., Nature 381 , 804-806, 1996) or CIA was expressed in mammalian cell lines. 293 cells were transfected. Lysates obtained by lysing cells 48 hours after transfection were subjected to immunoprecipitation using an anti-HA antibody in the same manner as in Example <4-1>. The immunocomplex kinase assay was performed to measure the SAPKβ activity of the immunoprecipitates obtained therefrom.

In addition, to investigate the effect of CIA on the expression of JNK1, pFR-Luc (Clonetech), pFA2-c-jun (Clonetech), pcDNA3-CIA, pcDNA3-ASK1 and pcDNA3-β-gal (Invitrogen) expressing Expression vectors were transiently transfected into 293 cells, a mammalian cell line. After 48 hours after transfection, the cells were hemolyzed and luciferase activity of the water-soluble fraction was analyzed using a luciferase assay kit (Promega). Luciferase activity of the transfected cells was analyzed based on the activity of β-galactosidase of the same cells.

As a result, ectopic expression of CIA also inhibited the enzyme activity and expression of ASK1, which also inhibited the kinase activity of JNK1 in the lower region of ASK1 ( FIG. 7 ). In addition, the transcriptional efficiency of c-Jun measured by luciferase activity was also significantly increased by ASK1 and inhibited as CIA coexpressed ( FIG. 8 ). Therefore, it can be seen that CIA also inhibits the expression and kinase activity of signal regulatory proteins located in the subregion of ASK1 through inhibition of ASK1 activity.

Example 5 CIA as a CAD-reactive protein

Caspase-activated deoxyribonuclease (CAD), a mouse counterpart of the human DFF40 protein, is known to play an important role in DNA fragmentation induced by apoptosis. Therefore, the present inventors have isolated the human fetus and the adult adult brain in the same manner as in Example <1-1> by a yeast two-hybrid screening method using a mouse CAD gene as a probe to separate a protein capable of binding to CAD. CDNA library was searched.

As a result, eight positive clones were selected for the transformants obtained from the cDNA library of the human fetal brain, and nine positive clones were selected for the transformants obtained from the cDNA library of the mouse adult brain. The clones were identified as clones having the same base sequence as CIA isolated as ASK1-reactive protein in the previous example. These results suggest that the CIA of the present invention, which acts as an inhibitor of ASK1 activity, can also physically react with CAD involved in DNA fragmentation to regulate apoptosis.

<Example 6> CAD binding capacity of the CIA protein

We have prepared GST-fusion recombinant proteins of various CAD to investigate which position of CIA protein binds to CAD.

Specifically, the wild type of CIA prepared in Example 4 and its deletion mutant, CIA-WT, CIA-ΔN, CIA-ΔC, and CIA-CEN recombinant protein were immobilized on glutathione-agarose beads. Post-in vitro transcribed [³⁵S] methionine-labeled CAD. On the other hand, wild-type CAD and CAD deletion mutants, full-length CAD-WT (1-345), N-terminal deleted CAD-ΔN (Δ1-83), CAD containing only the N-terminal half -NT (Δ162-345) and C-terminally deleted CAD-ΔC (Δ290-345) were prepared using PCR and cloned into pLexA vector (Clontech). CAD-WT, CAD-ΔN, CAD-NT and CAD-ΔC recombinant proteins isolated and purified from the E. coli transformants transformed with the expression vector³⁵After labeling with S radioisotope, GST-CIA was applied to immobilized glutathione-agarose beads. Proteins bound to each glutathione-agarose beads were eluted and then separated by SDS-polyacrylamide gel electrophoresis.

As a result, CIA-NT recombinant proteins comprising only full-length CIA-WT, C-terminal CIA-ΔC and N-terminal sites combined with ³⁵ S-labeled CAD physically in vitro transcribed, but with N CIA-ΔN deleted at the ends and CIA-CEN recombinant protein containing only the central site with deletion at both ends did not bind ³⁵ S-labeled CAD ( FIG. 9 ). In contrast, GST-fusion CIA proteins did not bind to ³⁵ S-labeled CAD in vitro. These results suggest that recombinant CIA proteins can bind directly to CAD and that the N-terminal region of CIA is involved in binding to CAD.

Generally, amino acid residues 1 to 83 of the CAD N-terminal region homologous to the conserved CIDE-N domain mediate binding to ICAD and residues 290 to 345 of the C-terminal region are It is known to be required for the nuclease activity of CAD. This was also confirmed by binding analysis using wild-type and deletion mutant recombinant proteins of CAD, GST-fusion CAD-WT, CAD-NT and C-terminal deletions containing only N-terminal half. -ΔC binds to the [ ³⁵ S] methionine-labeled CIA clone, but not the CAD-ΔN deleted N-terminus ( FIG. 9 ).

Therefore, CIA confirmed that the N-terminus of CIA specifically binds to the N-terminus of CAD, thereby inhibiting the activity of CAD.

Example 7 Inhibitory Effect of CIA on CAD Activity and DNA Fragmentation

<7-1> Inhibitory effect of CIA on in vitro CAD nuclease activity

In order to confirm the inhibitory effect of CIA on the in vitro CAD nuclease activity, His-CAD / T7-ICAD recombinant protein isolated and purified from Escherichia coli BL21 (DE3) was used. In order to isolate and purify the recombinant protein, the ICAD was cloned into the EcoRI / NotI restriction enzyme site of pET23b vector (Novagene), and then the CAD was cloned into the XbaI restriction enzyme site again, and the His-label and the ICAD Gene constructs displaying T7-labels at the C-terminus were prepared. The gene construct was transformed into E. coli BL21 (DE-3) (Pharmacia), and then E. coli transformants were cultured in the presence of 1 mM IPTG for 3 hours to induce the expression of the mixed protein. Recombinant protein produced from E. coli transformants was isolated using a NiNTA column (Quiagen) and used in the following experiment.

His-CAD / T7-ICAD recombinant protein (1 μg) isolated and purified as described above was added to each nuclease buffer solution (10 mM HEPES [pH 7.5] with or without the addition of recombinant caspase-3 (200 ng). ], Reaction with GST-fusion CIA or its deletion mutant recombinant protein (2 μg) dissolved in 1 mM EGTA, 5 mM MgCl ₂ , 50 mM NaCl, 1 mg / ml bovine serum albumin for 2 hours at 37 ° C. I was. After the reaction, the samples were electrophoresed on a 2% agarose gel and stained with ethidium bromide.

As a result, GST-CIA inhibited the nuclease activity of CAD recombinant protein and, in particular, inhibited CAD activity even in the presence of caspase-3, which reacts with CAD to induce nuclease activity of CAD. ICADL did not inhibit the nuclease activity of CAD even in the presence of caspase-3. In addition, in vitro nuclease analysis using recombinant proteins of wild-type and deletion mutants of CIA, GST-CIA-WT and GST-CIA-ΔC strongly inhibited the nuclease activity of CAD, but GST-CIA-ΔN And GST-CIA-CEN had no inhibitory effect ( FIG. 10 ). These results suggest that CIA inhibits the nuclease activity of CAD by directly binding to CAD through its N-terminus.

<7-2> Inhibitory Effect of CIA on DNA Fragmentation

To investigate the DNA fragmentation inhibition effect of CIA in mammalian cell line 293 cells, HA-labeled CIA clones or their deletion mutants were added with or without caspase-3 to 293 cells with CAD-Flag or ICADL. Co-expression was induced by transfection at the same time. After incubating the transfected cells for 48 hours, each chromosomal DNA was extracted and subjected to 2% agarose gel electrophoresis and stained with ethidium bromide.

As a result, overexpression of CAD / ICADL and caspase-3 in cells not simultaneously transfected with HA-CIA induced marked fragmentation of chromosomal DNA, whereas HA-CIA clones were transfected to co-express CIA. In cells, DNA fragmentation induced by CAD / ICADL and caspase-3 was inhibited ( FIG. 11 ). In addition, as a result of co-expression of the CIA deletion mutants into cells, DNA fragmentation activity of CAD was HA-CIA-ΔC, which is a mutant containing an N-terminal site involved in direct binding to CAD. It was inhibited by HA-CIA-CEN (FIG. 12).

Thus, CIA is an inhibitor of CAD that inhibits nuclease activity and DNA fragmentation activity of CAD by directly binding to CAD using the N-terminus of CIA.

Example 8 Reduction of Apoptosis and DNA Fragmentation Activity in 293-CIA Cells

<8-1> 293 Intracellular Transfection of CIA

As shown in the above examples, the inventors have found that CIA acts as an inhibitor of CAD binding ASK1 in n vitro to inhibit apoptosis and DNA fragmentation activity, and this CIA is actually anti- To examine whether it can act as an anti-apototic regulator, CIA clones were transfected to 293 cells, a mammalian cell line.

Specifically, 293 cells derived from human embryonic kidney cells were treated with DMEM containing 10% heat-inactivated fetal bovune serum and 100 U / ml penicillin / streptomycin (Gibco BRL). After inoculation on Dulbeco's modified Eagle's medium), the cells were incubated at 37 ° C. and 5% CO ₂ in a moisturizing incubator. In order to transfect HA-CIA by electrophoresis, cells of 1 × 10 ⁷ cells / plate concentration were lysed in 500 μl HEPES buffer and mixed with an appropriate amount of HA-CIA DNA. . The mixture was subjected to electroshock using Gene Pluser II (BioRad Inc. USA) according to the manufacturer's instructions, and the cells expressing HA-CIA were selected by culturing with G418. Expression of HA-CIA was examined by immunoblot analysis using monoclonal antibodies against HA from each colony selected.

As a result, as shown in FIG . 12 , HA-labeled CIA was stably expressed in 293 cells having low endogenous expression levels of CIA. The inventors performed the following experiment by selecting clone # 4 having the highest CIA expression level and clone # 6 having the lowest CIA expression level.

<8-2> Measurement of resistance to stress-induced apoptosis of 293-CIA cells

To investigate the effects of CIA on apoptotic apoptosis induced by various stresses in 293-CIA cells, 10 μg / ml cyclohexaxane was applied to 293 cells, 293-CIA # 4 cells and 293-CIA # 6 cells. Apoptosis was induced by treatment with 20 ng / ml TNF (TNF / CHX) or 1 μM staurosporine (STS) added with amide (cyclohexamide) or by irradiation with 80 J / m ² UV. Cells treated as above were incubated for 12 hours, 12 hours, or 24 hours, respectively, and then fixed for 1 hour by treating with 70% ethanol on ice. The immobilized cells were stained with 50 μg / ml popidium iodide (PI) and hypopoploid cell fractions were analyzed by FACS (FacsCalibur, Becton-Dickinson).

As a result, 293 cells and 293-CIA # 6 cells with low CIA expression levels were stimulated by apoptosis by TNF / CHX, STS, and UV light, resulting in a 5 to 8-fold increase in the total number of hypofloroid cell fractions compared to the control group. 293-CIA # 4 cells were relatively reduced in hypopoploid cell fractions by suppressing the stress-induced apoptosis due to the expression of CIA ( FIG. 13 ). Therefore, it was confirmed that the cells transfected with CIA of the present invention were resistant to apoptosis induced by stress stimulation.

<8-3> Measurement of resistance to ASK1-mediated apoptosis of 293-CIA cells

In Example <4-3>, it was confirmed that CIA inhibits apoptosis and DNA fragmentation by inactivating the ASK1-mediated JNK / SAPK pathway by inhibiting kinase activity of ASK1. Therefore, we investigated the expression vector pcDNA3-Daxx (498-740) in 293 and 293-CIA # 4 cells to investigate whether CIA actually inhibits ASK1 mediated apoptosis in cells. PCMV2-Flag-ΔMEKK1 (provided by Dr. Michael Karin, University of California, San Diego, USA) or pcDNA3-ASK1ΔN-Flag (PCR isolated ASK1 fragment from EcoRI / Xba I restriction enzyme of the pcDNA3-flag vector). Cloned to site) was transiently transfected by 2 μg. Transfected cells were incubated for 48 hours and then fixed in ice with 70% ethanol for 1 hour. The immobilized cells were stained with 50 μg / ml PI and hypopoploid cell fractions were analyzed by FACS.

As a result, ASK1-mediated apoptosis was activated by overexpression of Daxx in 293 cells transfected with Daxx-expressing expression vector, but the hypopoploid cell fraction increased, whereas CIA coexpressed in 293-CIA # 4 cells. Apoptosis was inhibited by inactivating the JNK / SAPK pathway, which was activated by the hypolipid cell fraction ( FIG. 14 ). These results suggest that CIA inhibits apoptosis by inhibiting ASK1 and hence JNK / SAPK activity as well as CAD-mediated DNA fragmentation.

The novel mouse CIA protein of the present invention and genes encoding it selectively bind CAD and ASK1 as negative regulators of apoptosis to inhibit apoptotic apoptosis and DNA fragmentation. It may be useful for selectively inhibiting proteins or treating diseases involving apoptosis-related processes, and the CAI gene may be used for gene therapy for such diseases.

Claims (8)

Mouse CIA (CAD inhibitor interacting with ASK1) gene, SEQ ID NO: 3 homologs or variants thereof. Mouse CIA protein set forth in SEQ ID NO: 4 or a variant thereof. 3. The variant protein of claim 2, wherein the variant protein is at least 90 partially in the portion corresponding to amino acid residues 1 to 79 of the N-terminal of SEQ ID NO: 4 and in the region corresponding to amino acid residues 174 to 221 of the C-terminal Mouse CIA protein or a variant protein thereof, characterized by having homology of not less than% and homology of not less than 80% as a whole. A nucleic acid or protein interacting with a CIA protein using the gene of claim 1 or the protein of claim 2 as a probe; Analogs of the CIA gene; Or searching for analogs of CIA proteins. The gene therapeutic agent comprising the gene construct containing the mouse CIA gene of Claim 1, its homologous gene, or its mutant gene as an active ingredient. ASK1 and CAD activity inhibitor comprising the mouse CIA protein of claim 2 or a mutant protein thereof as an active ingredient. Apoptosis-related disease treatment comprising the protein of claim 2. 8. The agent for treating apoptosis-related diseases according to claim 7, wherein the apoptosis-related disease is degenerative neurological disease, stroke, cancer, immune system disease or inflammation.